摘要:
建立同时测定大鼠肝微粒体中7种特异性底物对应代谢产物（对乙酰氨基酚、4″-羟基美芬妥英、右啡烷、4-羟基甲苯磺丁脲、6-羟基氯唑沙宗、1-羟基咪达唑仑和6β-羟基睾酮）的LC-MS/MS法，并应用探针底物法研究苯环喹溴铵对大鼠肝微粒体CYP450酶的抑制作用。将CYP450酶6种亚型的7种特异性探针底物非那西丁（CYP1A2）、S-美芬妥英（CYP2C11）、右美沙芬（CYP2D1/2）、甲苯磺丁脲（CYP2C6）、氯唑沙宗（CYP2E1）、咪达唑仑（CYP3A1/2）和睾酮（CYP3A1/2）分别与大鼠肝微粒体及系列浓度的苯环喹溴铵溶液进行温孵反应，合并处理后的微粒体溶液，采用LC-MS/MS法同时测定对应底物代谢产物的含量，计算相应的IC50，评价是否有抑制作用。并应用已知抑制剂对所建立的探针底物法进行验证。在大鼠肝微粒体温孵体系中，苯环喹溴铵对CYP1A2，CYP2C11，CYP2C6，CYP2E1和CYP3A1/2的IC50均大于100 μmol/L，对CYP2D1/2的IC50为（2.16±0.22）μmol/L。结果表明，苯环喹溴铵对CYP2D1/2可能有抑制作用，对CYP450酶的其他亚型无抑制作用。
Abstract:
To establish an HPLC-MS/MS methods for the simultaneous determination of seven metabolites (acetaminophen,4″-hydroxymephenytoin,dextrorphan,1-hydroxymidazolam,6β-hydroxyltestosterone,4-hydroxytolbutamide,6-hydroxychlorzoxazone) of CYP450 enzymes probe substrates in rat liver microsomes,and to investigate the inhibitory potency of bencycloquidium bromide on rat liver cytochrome P450 using probe substrates method.The probe substrate (phenacetin,S-mephenytoin,tolbutamide,dextromethorphan,chlorzoxazone,midazolam or testosterone) was incubated with RLM,bencycloquidium bromide or positive control,respectively.The precipitated samples for each probe substrate were mixed and simultaneous analyzed by LC-MS/MS.The concentrations of metabolites were assayed and used to calculate the IC50 which was used to evaluate the inhibition potency.The method was validated by incubating known CYP inhibitions -α-naphthoflavone (CYP1A2),omeprazole (CYP2C11),ketoconazole (CYP2C6,2E1,3A1/2),quinidine (CYP2D1/2) with individual probe substrate and rat liver microsomes,respectively.Positive controls show inhibition effects on each CYP 450s.The IC50 values of bencycloquidium bromide for CYP1A2,CYP2C11,CYP2C6,CYP2E1 and CYP3A1/2 were lower than 100 μmol/L,while the IC50 of bencycloquidium bromide for CYP2D1/2 was (2.16±0.22) μmol/L.It indicates that bencycloquidium bromid shows inhibitory effect on CYP2D1/2,but minor inhibitory effects of CYP1A2,CYP2C11,CYP2E1 and CYP3A1/2 in this study.
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